Nucleic acid probes for the detection and identification of fungi

ABSTRACT

Nucleic acid probes and primers are described for detecting fungi that cause disease in humans and animals, as well as spoilage of food and beverages. These probes can detect rRNA, rDNA or polymerase chain reaction products from a majority of fungi in clinical, environmental or food samples. Nucleic acid hybridization assay probes specific for Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Pseudallescheria boydii, and Sporothrix schenckii are also described.

FIELD OF INVENTION

The inventions described and claimed herein relate to the design andcomposition of two nucleic acid probes capable of detecting manydifferent fungal organisms in clinical, food, environmental and othersamples. The inventions described and claimed herein also relate to thedesign and composition of 21 probes capable of specifically detectingand identifying Aspergillus fumigatus, Blastomyces dermatitidis, Candidaalbicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum, Aspergillus flavus, Aspergillus glaucus, Aspergillus niger,Aspergillus terreus, Candida glabrata, Candida guilliermondii, Candidakerr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candidatropicalis, Pseudallescheria boydii, and Sporothrix schenckii inclinical, food, environmental and other samples.

BACKGROUND OF THE INVENTION

Fungi are eukaryotic microorganisms that are universally distributed.While in nature fungi play a major role in the decomposition of plantmaterials, they are also responsible for spoilage of food, beverage andpharmaceutical preparations. Out of an estimated 100,000 species offungi described by mycologists, approximately 150 species are pathogenicto man and animals. The increasing incidence of AIDS and the developmentof newer treatments for hematologic malignancies and organ transplantshas lead to an increase in the number of immunocompromised patients.These patients have a high risk of developing fungal infections, whichif not rapidly diagnosed and treated are capable of causing death in amatter of days. The number of antifungal drugs is limited and theirtoxic side effects on the patient are much higher than that ofcomparable antibacterial therapy. A rapid diagnosis of fungal infectionand start of treatment is critical in these patients. Books byKwon-Chung and Bennett, along with Sarosi and Davies, provide anoverview into the medical importance of fungi.

Fungal organisms are identified by morphology and nutritionalcharacteristics. Fungi may take anywhere from two days to several weeksto grow in culture and often the same organism can take radicallydifferent forms depending on the growth conditions. This makes timelyidentification difficult even for the classically trained expert andimpedes the treatment of patients where rapid identification of genusand species is of medical advantage.

The incidence and distribution of major pathogenic fungi varies bygeographic location. Aspergillus fumigatus, Blastomyces dermatitidis,Candida albicans, Coccidiodes immitis, Cryptococcus neoformans,Histoplaisma capsulatum, Paracoccidioides brasiliensis, Pseudallescheriaboydii and Sporothrix schenkii represent some of the leading causes ofmycotic infections.

Aspergillus fumigatus is among the top three causes of systemic fungalinfection treated in hospitals. It usually affects patients with organtransplants, acute leukemias and bums and can be rapidly fatal if notdiagnosed quickly. With over 150 species of Aspergillus present in thesoil, air and water, accurate detection of Aspergillus fumigatus becomesextremely important. Aspergillus clavatus, Aspergillus flavus,Aspergillus fumigatus, Aspergillus glaucus, Aspergillus nidulans,Aspergillus niger, Aspergillus ochraceus, Aspergillus terreus,Aspergillus unguis and Aspergillus ustus represent a majority ofAspergillus species seen in clinical specimens and their presence cancause diagnostic difficulties. Aspergillus flavus, Aspergillus fumigatusand Aspergillus niger have been linked with disease in humans, withAspergillus fumigatus being the predominant pathogen in North America. Afew immunologic tests exist for Aspergillus fumigatus but these havelimited sensitivity and specificity. There are also reports ofdevelopment of polymerase chain reaction based tests for Aspergillusfumigatus based on the amplification of the Asp fl antigen gene and aribosomal intergenic spacer (Spreadbury et. al.). The Spreadburytechnique is based on the PCR amplification of a 401 bp fragmentspanning the large subunit rRNA/mtergenic spacer region. This relies ona pair of primers to specifically amplify DNA from Aspergillus fumigatusonly, and is of no utility in identifying other fungi.

Blastomyces dermatitidis is present in the soil, usually in birddroppings and animal feces. Infections often occur at construction sitesand the ensuing lung infiltration and pneumonitis are usually fatal inimmunocompromised patients. Diagnosis by culture may take weeks, and theorganism is occasionally mistaken for other fungi. Existingimmunological diagnostic tests are unreliable, and there is a need forrapid and reliable DNA based diagnostic tests. Similarly, Histoplasmacapsulatum exists in the soil and is known to have infected at least 20%of the population of North America. Most infections start in the lungand resolve spontaneously, but may occasionally spread to other organs.AIDS patients represent a growing number of cases of Histoplasmosis.Diagnosis is difficult as immunological tests are often negative duringthe first 4-6 weeks of infection. Coccidioides immitis is found inabundance in the soil in Southwestern United States. Dust storms,farming, building construction, earthquakes and even hiking have beenlinked with outbreaks of disease. Lung infection followed by cavitationand disseminated miliary coccidioidomycosis are seen. Meningitis isusually lethal, and as with other fungi, mortality is highest indebilitated hosts. Four serotypes of Cryptococcus neoformans causedisease in humans. The incidence of this disease is growing rapidly,with up to 10% of HIV infected people developing cryptococcosis. DNAprobes capable of detecting all 4 serotypes are required for the earlydiagnosis and treatment for life threatening infections likecryptococcal meningitis. A report by Stockman et. al. discussescommercial tests for Histopkama, Blastomyces, Coccidioides, andCryptococcus based on the 18S rRNA (Gen-Probe, Inc., San Diego, Calif.).The authors report sensitivities ranging from 87.8 to 100% and aspecificity of 100%. One drawback of these probes is that these are usedon rRNA extracted from fungal cultures. As some fungi may require up to3 weeks to grow in culture, this technique cannot be used to expeditediagnosis until a culture becomes available.

Candida albicans is one of the most common causes of fungal infection inhumans. It is present in the respiratory, gastrointestinal and femalegenital tract of healthy individuals, and acts as an opportunisticpathogen in debilitated individuals on steroid or chemotherapy. Diabetesmellitus and indwelling catheters are other predisposing causes.Immunocompromised hosts show rapid hematogenous spread of fungi.Morbidity and mortality in untreated cases is high. Candida glabrata,Candida guilliernondii, Candida ken, Candida krusei, Candida lusitaniae,Candidaparapsilosis and Candida tropicalis are also known to causedisease in humans. DNA probes capable of identifying these individualspecies would eliminate the need for multiple blood cultures and lengthybiochemical speciation.

Recent advances in molecular techniques have led to the approach ofmicrobe detection and identification based upon the DNA sequence ofribosomal genes. Commonly used detection techniques include eitherdirect amplification of the ribosomal DNA (rDNA) genes by the polymerasechain reaction, or reverse transcription of the ribosomal RNA (rRNA)into complementary DNA (cDNA) followed by polymerase chain reactionamplification of the cDNA. Ribosomes are composites of unique rRNA andprotein species that function in the translation of messenger RNA intoprotein. Evolutionary studies are consistent with the interpretationthat all extant life has evolved from a single organism. Thus, allcellular organisms contain rRNA and these rRNAs are related byevolution. The evolutionary process is such that each species oforganism appears to have unique regions of sequence in its ribosomalgenes. The presence of these unique species specific regions allows oneto design DNA probes that under conditions of hybridization willspecifically bind to, and identify the polymerase chain reactionamplified DNA from only one species of fungus. For the purposes of thisapplication, the word "primer" is used to mean a nucleotide sequencewhich can be extended by template-directed polymerization, and "probe"is used to mean a nucleotide sequence capable of detecting itscomplementary sequence by hybridization. Also, for the purpose of thisapplication, the phrase "nucleotide sequence" is intended to includeeither DNA or RNA forms or modification thereof. Furthermore, thoseversed in the art will recognize that primer sequences can be used asprobes and vice versa. The use of nucleic acid hybridization to detectspecific nucleic acid sequences of interest is also described by Kohne(U.S. Pat. No. 4,851,330, 7/1989).

In prokaryotes and eukaryotes, ribosomal RNA and the corresponding rDNAgenes are identified by the size of the RNA. The sizes are related interms of sedimentation velocity or S values. Thus, for prokaryotes thevalues are 5S, 16S, and 23S; and for eukaryotes the values are 5S, 5.8S,18S and 28S. Because all ribosomes perform the same function which isessential for cell viability, ribosomal sequences are largely conserved,yet certain regions of each ribosomal species are subject to morevariation without consequence to function. It is these hypervariableregions that allow one to identify different species amongst members ofthe same genus. As noted in the referenes, there are several reportswhere 5S, 18S and the intergenic spacer between 5.8S and 28S rDNA havebeen used for the detection and identification of fungi (Holmes et. al.,Hopfer et. al., Lott et. al., Maiwald et. al., Makimura et. al.,Mitchell et. al., Nakamura et. al.). Holmes et. al. describe a PCR testbased on the co-amplification of the 5S rDNA and an adjacentnontranscribed spacer region. This identifies only Candida albicans anddetects other Candida species without identifying individual organisms.Hopfer et. al. and Maiwald et. al. both use universal primers to amplify18S rDNA from several fungi including Candida sp., Aspergillusfumigatus, Cryptococcus neoformans and Trichosporon sp. These ampliconsare digested with restriction enzymes and the cut fragments are sized bygel electrophoresis. This restriction fragment length polymorphismpattern enables them to identify most but not all organisms. Thistechnique can be used on amplified DNA from a pure fungal culture. Asclinical samples such as sputum usually contain multiple fungalorganisms, this technique has little utility in diagnosis as multipleoverlapping fragments obtained from a mix of fungi would be nearlyimpossible to interpret. Lott et. al. use the 5.8S RNA and the internaltranscribed spacer (ITS2) to identify and speciate Candida albicans andrelated Candida species. Makimura amplifies a 687 bp fragment from the18S rDNA of 25 medically important fungi and uses these in the diagnosisof Candida albicans in clinical samples. Mitchell uses nested PCR toamplify 5.8S and internal transcribed spacer (ITS) to identifyCryptococcus neoformans. No subsequent testing is done to verify theidentity of the amplified DNA. Nakamura et. al. use 18S primers todetect Aspergillus fumigatus infections of the lung. Most protocolsgiven in these references can only be used to detect an extremelylimited number of fungi from a clinical specimen. Hopfer et. al. andMaiwald et. al. can identify multiple organisms from pure cultures, buttheir utility for clinical specimens containing multiple fungal speciesis limited at best.

United States patents have been issued to Weisburg et. al. for probesdeveloped for the detection of 18S small subunit ribosomal RNA sequencesin fungi. These probes will detect fungi from many species, but cannotbe used easily to identify any single species. United States patentshave also been issued to Milliman for probes developed for the specificdetection of the bacteria Staphylococcus aureus based on the 23Sribosomal sequence (U.S. Pat. No. 5,292,874) and Histoplasma capsulatumbased on the 18S ribosomal sequence (U.S. Pat. No. 5,352,579). Hogan et.al. (European Pat. App. 0,272,009) describe one fungal probe for 18SrRNA and three fungal probes for 28S rRNA sequences. None of the 28Sprobes described by Hogan et. al. is related to any of the probesdescribed in our invention. All probes claimed in our invention can bemapped within the first 900 base pairs of a 28S gene. The probesdescribed by Hogan et. al. are located further 3' on the 28S sequence,between base pairs 1000 and 2000 (these numbers are comparable to theprimary sequence of Saccharomyces cerevisiae 28S rRNA gene. Genbankaccession number: J01355). Leclerc et. al. have published reportsanalyzing the phylogenetic relationship between fungi based on partialDNA sequences of several fungal 28S genes sequenced by them. Some of theorganisms claimed to have been sequenced by Leclerc are the same as someorganisms sequenced by us. These are Sporothrix schenckii,Pseudallescheria boydii, Blastomyces dermatitidis, Histoplasmacapsulatum and Chrysosporium sp. Leclerc et. al. have not published anysequence data in their report, and to the best of our knowledge, thesesequences have not been made available in the GenBank. Thereverse-complement sequence of their sequencing primer 401 (TCCCTTTCAACAATTTCACG) overlaps our SEQ ID NO: 1 (GTGAAATTGT TGAAAGGGAA) by 19nucleotides and their sequencing primer 636 (GGTCCGTGTT TCAAGACGG)overlaps our SEQ ID NO: 2 (GACTCCTTGG TCCGTGTT) by 10 nucleotides. Weare aware of no reports in the literature of variable regions from 28SrRNA genes of fungi being used as targets for the development of speciesspecific diagnostic probes.

As discussed above, most present techniques for the molecular detectionof fungi rely on the use of highly specific primers for the PCRamplification of only one fungal species. Those that employ "Universal"primers for a PCR amplification of DNA from multiple organisms, usepost-PCR amplicon identification techniques that are useful only on purecultures of fungi. These are not able to identify fungi from a clinicalspecimen containing multiple fungal organisms. Our first aim was todevelop "Universal" primers for the 28S gene. These primers would becapable of amplifying in a PCR, 28S rDNA from most fungi. Our subsequentaim was to develop species specific probes for fungi of interest, thatwould be used to analyze our "Universal" 28S amplicon. These speciesspecific probes would be able to detect the presence of fungi ofinterest even in situations containing mixed fungal species.

One aspect of this invention is to provide nucleic acid primers capableof detecting 28S sequences from DNA or RNA of most fungi. These would beused as "Universal" primers in a polymerase chain reaction to amplify28S sequences from any fungus present in clinical, food, environmentalor other samples.

A second aspect of this invention is to provide nucleic acid probescapable of detecting and identifying, by nucleic acid hybridization, thepathogens Aspergillus fumigatus, Blastomyces dermatitidis, Candidaalbicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasmacapsulatum, Aspergillus flavus, Aspergillus glaucus, Aspergillus niger,Aspergillus terreus, Candida glabrata, Candida guilliermondii, Candidakefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candidatropicalis, Pseudallescheria boydii, and Sporothrix schenckii by use ofany of several different formats.

SUMMARY OF THE INVENTION

Nucleic acid probes and primers are described for detecting fungi thatcause disease in humans and animals, as well as spoilage of food andbeverages. These probes can detect rRNA, rDNA or polymerase chainreaction products from a majority of fungi in clinical, environmental orfood samples. Nucleic acid hybridization assay probes specific forAspergillus fumigatus, Blastomyces dermatitidis, Candida albicans,Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum,Aspergillus flavus, Aspergillus glaucus, Aspergillus niger, Aspergillusterreus, Candida glabrata, Candida guilliermondii, Candida kefyr,Candida krusei, Candida lusitaniae, Candida parapsilosis, Candidatropicalis, Pseudallescheria boydii, and Sporothrix schenckii are alsodescribed.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 represents the relative position of the sequences described onthe 28S subunit of fungi.

DETAILS OF THE INVENTION

Our first objective was to develop nucleic acid primers for use in apolymerase chain reaction to amplify 28S genes from all fungi likely tobe present in a clinical sample. This amplified DNA would then beamenable to probing with several different species specific probes. Eachone of these species specific probes would, under conditions ofhybridization, anneal to 28S ribosomal DNA from only one species offungus, thereby detecting and identifying the species of fungus presentin the clinical sample. The 28S gene was selected as a target because ithad regions that were conserved among fungi and these would providepotential annealing sites for "universal" fungal probes. The ribosomal28S genes were also expected to have hypervariable regions that would beunique enough to provide sites for species specific probes. The largerRNA gene is called the 23S rRNA gene in prokaryotes and 28S ineukaryotes. This designation is based on the length and therefore thesedimentation coefficient of these rRNA molecules. Fungal large subunitrRNAs vary in size among different organisms and are often referred toas being 25S, 26S or 28S. Since fungi are eukaryotes, and to maintainuniformity in this application, we shall refer to fungal large subunitrRNA as 28S rRNA.

Published sequences from Cryptococcus neoformans, two Candida albicans,Saccharomyces cerevisiae and two Schizosaccharomyces pombe 28S genes areapproximately 3.5 kilobases in length (Genbank accession numbers:L14068, L28817, X70659, J01355, Z19136 & Z19578). These four sequenceswere aligned, and a region of sequence variability was found clusteredbetween coordinates 200 and 700 from the 5' end of these genes. As aninitial starting point, two nucleic acid primers P1 (ATCAATAAGCGGAGGAAAAG) SEQ ID NO:24 and P2 (CTCTGGCTTC ACCCTATTC) SEQ ID NO:25 (seeFIG. 1), capable of hybridizing to all 4 of the above mentionedorganisms and not to human 28S sequences (GenBank accession number:M11167), were designed and used under low stringency hybridizationconditions in a polymerase chain reaction to amplify approximately 800base pairs of DNA spanning this hypervariable region from the following34 fungi that were obtained from the Mayo Clinic fungal collection:Acremonium sp., Aspergillus clavatus, Aspergillus fumigatus, Aspergillusglaucus, Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus,Aspergillus terreus, Aspergillus unguis, Aspergillus ustus, Beauvariasp., Bipolaris sp., Blastomyces dermatitidis, Candida albicans, Candidaglabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candidalusitaniae, Candidaparapsilosis, Candida tropicalis, Chrysosporium sp.,Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var.neoformans, Curvularia sp., Geotrichum sp., Histoplasma capsulatum,Mucor sp., Penicillium sp., Pseudallescheria boydii, Saccharomycescerevisiae, Sporothrix schenkii and Trichosporon beigelii.

DNA was extracted from the fungi listed above by the following method. Aloopful of fungal culture was scraped off a culture plate using asterile inoculation loop. The fungus was added one milliliter of sterilewater in a 1.5 ml Sarsted (Newton, N.C.) screw cap microcentrifuge tube.This tube was placed in a boiling water bath for 20 minutes in order tolyse the fungus and release DNA from the cells. Two microliters of thiswhole cell lysate was used in a PCR to amplify 28S rDNA. All PCRamplifications were carried out as hot-start reactions in a 50 ulreaction volume using Perkin-Elmer (Norwalk, Conn.) 0.5 ml thin-wallpolypropylene tubes and a Perkin-Elmer thermal cycler. Reagents added tothe tube initially were 2.5 ul of 10×PCR buffer (100 mM tris pH 8.3, 500mM KCl, 15 mM MgCl₂), 5.0 ul of 50% glycerol/1 mM cresol red, 8.0 ul ofdNTP mix (1.25 mM each of dATP, dGTP, dTTP and dCTP), 12 picomoles ofeach nucleic acid primer and sterile water to make up a volume of 25 ul.A wax bead (Ampliwax Gem-100, Perkin-Elmer) was added and the tubesheated to 77° C. for 1 minute and cooled to room temperature to form awax barrier. 2.5 ul of 10×PCR buffer, 5.0 ul of 50% glycerol/1 mM cresolred, 0.2 ul Taq polymerase (AmpliTaq 5 U/ul, Perkin-Elmer) and 15.3 ulof sterile water was added to the tube along with 2.0 ul of DNA from thefungal whole cell lysate described above. 50 cycles of thermal cyclingwas carried out at 94° C.--30 sec, 40° C.--1 min, 72° C.--2 min. Theamplified DNA was purified from a low melt agarose gel by tris bufferedphenol pH 8.0, phenol/chloroformlisoamyl alcohol (25:24:1 by vol.) and 3ether extractions, followed by isopropanol precipitation and 70% ethanolwash.

We completely sequenced both strands of DNA amplified from the organismslisted above. All sequencing was carried out on an Applied Biosystems373A sequencer. Every nucleotide in the sequences generated was verifiedand confirmed by examining the complementary nucleotide from the secondstrand sequence. We had now created a novel database consisting ofnucleic acid sequences spanning a variable region of the 28S rDNA from adiverse collection of medically important fungi.

While the complete sequences for Candida albicans, Cryptococcusneoformans and Saccharomyces cerevisiae 28S genes had previously beenpublished and deposited in GenBank, it was not obvious, nor had it beendefined, whether any regions of sequence identity among these threeorganisms would also be conserved among all fungi of interest. DNAsequences from all the fungi in our novel 28S database had to beanalyzed in order to develop "Universal" 28S probes. All sequences weresubjected to extensive manipulation to identify optimal relativealignments in order to identify regions of similarity for use as"Universal" probes. The selected probe sequences had to meet severalimportant criteria besides the condition of being present in 28S genesfrom most fungal species. Each probe sequence required an appropriatethermal profile, secondary structure and utility in a DNA amplificationreaction. These probes were optimized to work for PCR amplification inpure cultures of fungus, as well as in the presence of DNA from multiplesources as in the case of clinical specimens. The probes were alsodesigned to facilitate direct sequencing of the amplified DNA. Ouranalysis led to the discovery of the oligonucleotide probes listed in(SEQ ID NO: 1) and (SEQ ID NO:2). (For their location, see FIG. 1.) Thesuccessful identification of these two probes ((SEQ ID NO: 1) and (SEQID NO:2)) completed our first objective to develop nucleic acid probesthat would hybridize to, and detect 28S rRNA and rDNA from a majority offungi (FIG. 1 and Table 1). As shown later in this application, thenovel sequence information generated by the use of our "Universal"probes allowed us to develop species-specific probes ((SEQ ID NO:3) to(SEQ ID NO:23)) capable of identifying 19 different disease-causingfungi.

                  TABLE 1                                                         ______________________________________                                        Presence of hybridization sites for probes SEQ ID NO: 1 and SEQ ID NO:        2 in 28S nucleic acid sequences.                                                                SEQ ID NO: 1                                                                             SEQ ID NO: 2                                     ______________________________________                                        Acremonium sp.    +          +                                                Aspergillus clavatus                                                                            +          +                                                Aspergillus flavus                                                                              +          +                                                Aspergillus fumigatus                                                                           +          +                                                Aspergillus glaucus                                                                             +          +                                                Aspergillus nidulans                                                                            +          +                                                Aspergillus niger +          +                                                Aspergilius ochraceus                                                                           +          +                                                Aspergillus terreus                                                                             +          +                                                Aspergillus unguis                                                                              +          +                                                Aspergillus ustus +          +                                                Aspergillus sp.   +          +                                                Beauvaria sp      +          +                                                Bipolaris sp.     +          +                                                Blastomyces dermatitidis                                                                        +          +                                                Blastoschizomyces sp.                                                                           +          +                                                Candida albicans  +          +                                                Candida glabrata  +          +                                                Candida guilliermondii                                                                          +          +                                                Candida kefyr     +          +                                                Candida krusei    +          +                                                Candida lusitaniae                                                                              +          +                                                Candida parapsilosis                                                                            +          +                                                Candida tropicalis                                                                              +          +                                                Chrysosporium sp. +          +                                                Cladosporium sp.  +          +                                                Coccidioides immitis                                                                            +          +                                                Cryptococcus laurentii                                                                          +          +                                                Cryptococcus neoformans                                                                         +          +                                                Cryptococcus neoformans var                                                                     +          +                                                gattii                                                                        Cryptococcus terreus                                                                            +          +                                                Curvularia sp     +          +                                                Filobasidiella neoformans                                                                       +          +                                                Filobasidium capsuligenum                                                                       +          +                                                Filobasidium uniguttalatum                                                                      +          +                                                Fusarium sp.      +          +                                                Geotrichum sp.    +          +                                                Histoplasma capsulatum                                                                          +          +                                                Malbranchea sp.   +          +                                                Mucor sp.         +          +                                                Paecilomyces sp.  +          +                                                Penicillium sp.   +          +                                                Pseudallescheria boydii                                                                         +          +                                                Rhizopus sp.      +          +                                                Saccharomyces cerevisiae                                                                        +          +                                                Scopulariopsis brevicaulis                                                                      +          +                                                Scopulariopsis brumptii                                                                         +          +                                                Sporothrix schenckii                                                                            +          +                                                Trichosporon beigelii                                                                           +          +                                                Human             -          +                                                ______________________________________                                    

Probes SEQ ID NO: 1 and SEQ ID NO: 2 were used to successfully amplify(Table 2) and sequence DNA spanning this variable region from thefollowing 49 organisms: Acremonium sp., Aspergillus clavatus,Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus,Aspergillus nidulans, Aspergillus niger, Aspergillus ochraceus,Aspergillus sp., Aspergillus terreus, Aspergillus unguis, Aspergillusustus, Beauvaria sp., Bipolaris sp., Blastomyces dermatitidis,Blastoschizomyces sp., Candida albicans, Candida glabrata, Candidaguilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, Chrysosporium sp.,Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var.neoformans, Cryptococcus neoformans var. gattii, Cryptococcus terreus,Cryptococcus laurentii, Curvularia sp., Filobasidiella neoformans,Filobasidium capsuligenum, Filobasidium uniguttulatum, Fusarium sp.,Geotrichum sp., Histoplasma capsulatum, Malbranchea sp., Mucor sp.,Paecilomyces sp., Penicillium sp., Pseudallescheria boydii, Rhizopussp., Saccharomyces cerevisiae, Scopulariopsis brevicaulis,Scopulariopsis brumptii, Sporothrix schenkii and Trichosporon beigelii.This sequence information generated by the use of probes SEQ ID NO: 1and SEQ ID NO: 2 expanded the size of our database consisting of fungal28S sequences. All amplified DNA was sequenced across both strands froma minimum of two different isolates of each organism to ensure accuracyof the data generated.

                  TABLE 2                                                         ______________________________________                                        Polymerase chain reaction amplification of 28S rDNA with probes SEQ           ID NO: 1 and SEQ ID NO: 2.                                                                      PCR with SEQ ID                                                               NO: 1 & NO: 2                                               ______________________________________                                        Acremonium sp.    +                                                           Aspergillus clavatus                                                                            +                                                           Aspergillus flavus                                                                              +                                                           Aspergillus fumigatus                                                                           +                                                           Aspergillus glaucus                                                                             +                                                           Aspergillus nidulans                                                                            +                                                           Aspergillus niger +                                                           Aspergillus ochraceus                                                                           +                                                           Aspergillus terreus                                                                             +                                                           Aspergillus unguis                                                                              +                                                           Aspergillus ustus +                                                           Aspergillus sp.   +                                                           Beauvaria sp.     +                                                           Bipolaris sp.     +                                                           Blastomyces dermatitidis                                                                        +                                                           Blastoschizomyces sp.                                                                           +                                                           Candida albicans  +                                                           Candida glabrata  +                                                           Candida guilliermondii                                                                          +                                                           Candida kefyr     +                                                           Candida krusei    +                                                           Candida lusitaniae                                                                              +                                                           Candida parapsilosis                                                                            +                                                           Candida tropicalis                                                                              +                                                           Chrysosporium sp. +                                                           Cladosporium sp.  +                                                           Coccidioides immitis                                                                            +                                                           Cryptococcus laurentii                                                                          +                                                           Cryptococcus neoformans                                                                         +                                                           Cryptococcus neoformans var                                                                     +                                                           gattii                                                                        Cryptococcus terreus                                                                            +                                                           Curvularia sp.    +                                                           Filobasidiella neoformans                                                                       +                                                           Filobasidium capsuligenum                                                                       +                                                           Filobasidium uniguttulatum                                                                      +                                                           Fusarium sp.      +                                                           Geotrichum sp.    +                                                           Histoplasma capsulatum                                                                          +                                                           Malbranchea sp.   +                                                           Mucor sp          +                                                           Paecilomyces sp.  +                                                           Penicillium sp.   +                                                           Pseudallescheria boydii                                                                         +                                                           Rhizopus sp.      +                                                           Saccharomyces cerevisiae                                                                        +                                                           Scopulariopsis brevicaulis                                                                      +                                                           Scopulariopsis brumptii                                                                         +                                                           Sporothrix schenckii                                                                            +                                                           Trichosporon beigelii                                                                           +                                                           Human             -                                                           ______________________________________                                    

This list of fungi sequenced by us represents organisms responsible formost cases of subcutaneous and deep mycotic infections in humans andalso includes saprophytes (non-pathogenic fungi) commonly encountered inclinical isolates. Since the two probes (SEQ ID NO: 1 and SEQ ID NO: 2)hybridize to 28S rDNA from all the fungi listed above, they are capableof diagnosing the presence of a majority of fungi that are likely to bepresent in a clinical specimen. They are believed to be primers foruniversally detecting fungi.

Probes listed in SEQ ID NO: 1 and SEQ ID NO: 2 were also checked fortheir potential ability to hybridize to, and amplify (in a polymerasechain reaction) 23 S sequences from bacteria by searching forhybridization sites among the 539 bacterial 23S genes listed in GenBank.Bacterial 23S rDNAs do not have suitable hybridization sites for SEQ IDNO: 1 and SEQ ID NO: 2 and these two probes should not be able toamplify bacterial DNA under stringent conditions.

Our second objective was to develop species specific probes, which underhybridization conditions, would detect Aspergillus fumigatus,Blastomyces dermatitidis, Candida albicans, Coccidioides immitis,Cryptococcus neoformans, Histoplasma capsulatum, Aspergillus flavus,Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Candidaglabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candidalusitaniae, Candida parapsilosis, Candida tropicalis, Pseudallescheriaboydii, and Sporothrix schenckii. We used our database of fungal 28Snucleic acid sequences to create a multiple sequence alignment of allthe organisms that we had sequenced. Every individual sequence wassubjected to intensive comparison with all other sequences in ourdatabase in order to discover unique regions of sequence that would bepresent only in the fungus of interest, and would be absent in all otherfungi. When unique stretches of sequence were identified, these werefurther analyzed for thermal profile and secondary structure. Each probeconstructed by us will, under conditions of hybridization, specificallyhybridize to and detect, nucleic acid sequence from the unique region ofonly one specific target fungus. Those versed in the art will recognizethat specification of a single-stranded DNA sequence implies the utilityof the complementary DNA sequence, as well as the two equivalent RNAsequences. Furthermore, sequences incorporating modification of any ofthe moieties comprising the nucleic acid (i.e., the base, the sugar orthe backbone) are functional equivalents of the sequence. It should alsobe recognized that these additional sequences can potentially serve asprobes or primers.

The nucleic acid sequences for these species specific synthetic probesare listed in SEQ ID NO: 3 to SEQ ID NO: 23. There are two probesspecific for Cryptococcus neoformans, two probes specific for Sporothrixschenckii, and one probe each for Aspergillus fumigatus, Blastomycesdermatitidis, Candida albicans, Coccidioides immitis, Histoplasmacapsulatum, Aspergillus flavus, Aspergillus glaucus, Aspergillus niger,Aspergillus terreus, Candida glabrata, Candida guilliermondii, Candidakefyr, Candida Armsei, Candida lusitaniae, Candida parapsilosis, Candidatropicalis and Pseudallescheria boydii 28S rRNA and rDNA. (See Tables3-6 and further discussion below.)

All species specific probes developed by us are novel and to the best ofour knowledge have not been reported in the literature. While all 28Sgenes sequenced by us had several regions that were different among thevarious species analyzed, the regions that would function best asspecies specific probes under conditions of hybridization were notobvious. Extensive analysis of each 28S sequence yielded severalpotential probe sites. These were studied in detail to enable theselection of optimal unique sites for each probe, based on the need toobtain optimal hybridization characteristics under the test conditions.The highly specific hybridization characteristics of all probe sequencesdeveloped by us were then validated by experimental results. The priorexistence in GenBank of sequences for Candida albicans and Cryptococcusneoformans 28S genes was in itself not sufficient to enable even anindividual versed in this field to develop specific probes for either ofthese two organisms. We had to obtain novel 28S sequence from Candidaalbicans, Candida glabrata, Candida guilliernondii, Candida kefyr,Candida krusei, Candida lusitaniae, Candida parapsilosis, Candidatropicalis, Cryptococcus neoformans var. neoformans, Cryptococcusneoformans var. gatti, Cryptococcus terreus, Cryptococcus laurentii,Filobasidiella neoformans, Filobasidium capsuligenum and Filobasidiumuniguttulatum before we were able to identify potential regions for thedevelopment of species specific probes for these two fungal organismsthat would not cross react with the others listed above.

Our modification of the Chomczynski technique (see Example 2, below)allows us to obtain DNA from any clinical specimen, irrespective ofsource (see Table 8 for a variety of clinical specimens tested), withina 3 hour period. The PCR amplification and subsequent probing can beaccomplished with ease within a 24 hour period. The final identificationis therefore possible in a day as opposed to several days or weeksrequired by traditional methods. This speed and sensitivity of diagnosiscan make a difference between life and death in debilitated patientsbattling fungal diseases of undetermined cause. Rapid diagnosis willallow physicians to immediately direct their therapy towards curing theidentified causative fungus, rather than wait for days or weeks whilethe patient succumbs to an unknown fungus.

Our probes have the ability to pick out the correct target organism evenin a mixed fungal infection because of their high level of specificity.The methods of Hopfer et. al. and Maiwald et. al., do not allowidentification of individual species in a mixed fungal infection becauserestriction fragment length polymorphism results are nearly impossibleto interpret when multiple organisms contribute to the restrictionfragments. Their method can therefore only be used on a pure culture,and this also does not save any diagnostic time, because the fungusfirst has to be grown in culture.

The probes developed by us allow rapid species identification of a largenumber of pathogenic fungi by using multiple probes against only one PCRamplified fragment of DNA. Coupled with our modified DNA extractiontechnique and our ability to accurately diagnose in the case of mixedorganisms, this strategy can provide the greatest amount of diagnosticinformation in the shortest amount of time. This diagnostic strategy isalso amenable to automation, which can result in even greater savings intime, money and effort.

The sequences and the complement of the sequences claimed in thisdisclosure, along with any modifications to these sequences, maypotentially be utilized in assays for the identification of fungi basedon several existing methodologies, as well as future improvements andalterations of this technology. These techniques include, but are notlimited to, assays based on hybridization, ligation, polymerization,depolymerization, sequencing, chemical degradation, enzymatic digestion,electrophoresis, chromatography and amplification. Furthermore, all suchvariations ultimately are based in some selection or amplificationprocess, some ligand or some nucleic acid moiety that recognizes orutilizes the sequences (SEQ ID NO: 1) to (SEQ ID NO:23) claimed in thisapplication. Such variations include but are not limited to use of avariety of linear or exponential target amplification schemes, such as,any of the myriad forms of PCR, the ligase chain reaction, Q-betarepliase, etc.; direct detection of species-specific nucleic acidpurified or extracted from pure fungal culture using a probe selectedfrom the group (SEQ ID NO: 3) to (SEQ ID NO: 23); use of thecomplementary DNA forms of (SEQ ID NO: 1) to (SEQ ID NO:23); use of theRNA forms of these sequences and their complements; and use ofderivatives of these DNA or RNA sequences by the addition of one or moremoieties from a variety of labels including nucleic acid sequences,proteins, signal generating ligands such as acridinium esters, and/orparamagnetic particles. These techniques may be utilized with DNA, RNAor modified derivatives used as either the target or the detectionmolecule.

Further variations of the invention that utilize any of the namedsequences will be apparent to those with ordinary skill in the art. Thefollowing examples illustrate various aspects of the invention but arenot intended to limit its usefulness.

EXAMPLE 1.

Testing probes SEQ ID NO: 3 to SEQ ID NO: 23 for hybridizationspecificity.

Probes listed in SEQ ID NO: 3 to SEQ ID NO: 23 were tested forspecificity against their target organisms. Probe SEQ ID NO: 5 forCandida albicans was the first one tested against a panel of fungi takenfrom the Mayo Clinic collection. 28S rDNA from Acremonium sp.,Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus,Aspergillus glaucus, Aspergillus nidulans, Aspergillus niger,Aspergillus ochraceus, Aspergillus terreus, Aspergillus unguis,Aspergillus ustus, Aspergillus sp., Beauvaria sp., Bipolaris sp.,Blastomyces dermatitidis, Candida albicans, Candida glabrata, Candidaguilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae,Candida parapsilosis, Candida tropicalis, Chrysosporium sp.,Cladosporium sp., Coccidioides immitis, Cryptococcus neoformans var.neoformans, Curvularia sp., Fusarium sp., Geotrichum sp., Histoplasmnacapsulatum, Mucor sp., Penicillium sp., Pseudallescheria boydii,Rhizopus sp., Saccharomyces cerevisiae, Scopulariopsis breviacaulis,Sprothrix schenkii and Trichosporon beigelii was amplified in apolymerase chain reaction using oligonucleotide probes SEQ ID NO: 1 andSEQ ID NO: 2. All PCR amplifications were carried out as hot-startreactions in a 50 ul reaction volume using Perkin-Elmer (Norwalk, Conn.)0.5 ml thin-wall polypropylene tubes and a Perkin-Elmer thermal cycler.Reagents added to the tube initially were 2.5 ul of 10×PCR buffer (100mM tris pH 8.3, 500 mM KCl, 15 mM MgCl₂), 5.0 ul of 50% glycerol/1 mMcresol red, 8.0 ul of dNTP mix (1.25 mM each of dATP, dGTP, dTTP anddCTP), 11 picomoles of each nucleic acid primer and sterile water tomake up a volume of 25 ul. A wax bead (Ampliwax Gem=100, Perkin-Elmer)was added and the tubes heated to 77° C. for 30 seconds and cooled toroom temperature to form a wax barrier. 2.5 ul of 10×PCR buffer, 5.0 ulof 50% glyceroyl/1 mM cresol red, 0.2 ul Taq polymerase (AmpliTaq 5U/ul, Perkin-Elmer) and 15.3 ul of sterile water was added to the tubealong with 2.0 ul of DNA from the fungal whole cell boiled lysatedescribed above. 50 cycles of thermal cycling was carried out at 94°C.--30 sec, 50° C.--1 min, 72° C.--2 min. Five microliters of polymerasechain reaction mix from each sample was run on a 5% polyacrylamide gelto visually confirm the successful amplification of 28S rDNA from eachfungus listed above. 40 ul of the remaining amplified 28S rDNA wasdenatured in 1 N NaOH, and half of this denatured rDNA was slot blottedon to a positively charged polysulphone based membrane equilibrated in0.5N NaOH. The membrane was air dried for 15 minutes and baked in avacuum oven at 80° C. for 30 minutes. Amplified rDNA from each specieswas now bound and immobilized at a separate spot on the membrane. Thefree binding sites on the membrane were blocked by incubating themembrane for 3 hours at 40° C. in hybridization buffer (100 ml ofhybridization buffer was made using 1 g non-fat milk powder, 6 g NaH₂PO₄, 7 g SDS, 200 ul 0.5M EDTA and adjusted to pH 7.2 with NaOH). Thespecific probe for Candida albicans (SEQ ID NO: 5) was end-labeled withradioactive phosphorus using ³² P ATP and T4 polynucleotide kinase. 50picomoles of this probe was added to 70 milliliters of hybridizationbuffer and the membrane was probed at 40° C. overnight. The membrane waswashed in hybridization buffer at 40° C. for 15 minutes followed by awash in 2×SSC at 40° C. for 15 minutes. The membrane was then exposed onx-ray film for at least 1 hour. The oligonucleotide probe SEQ ID NO: 5only hybridized to amplified 28S rDNA from Candida albicans (see Table3) Under these hybridization conditions, probe SEQ ID NO: 5 is extremelyspecific for Candida albicans. The sequence of oligonucleotide probe SEQID NO: 5 differs from the sequences of other species of Candida by asfew as 1 or 2 bases, but these mismatches are sufficient to preventstable hybrids from forming with the other Candida species.

Probes SEQ ID NO: 3 to SEQ ID NO: 23 were tested for specificity, asdescribed above for the Candida albicans probe SEQ ID NO: 5, against thesame panel of fungi listed in the preceding paragraph. The positivelycharged polysulphone based membrane probed with Candida albicans probeSEQ ID NO: 5 was washed in 0.5N NaOH at 40° C. for 10 minutes to removeall bound Candida albicans probe. The membrane was sequentially probedwith all probes listed in SEQ ID NO: 3 to SEQ ID NO: 23. For eachsubsequently tested probe, the membrane was blocked for at least 30minutes, probe hybridization was carried out at 40°-42° C. for at least3 hours, and post-hybridization washes were done in 2×SSC for 20minutes. The membrane was stripped between probings by washing in 0.5 to1.0N NaOH at 40°-42° C. Results are listed in Tables 3 to 6.

As shown in Tables 3 to 6, each probe listed in SEQ ID NO: 3 to SEQ IDNO: 23 specifically hybridizes to only one target fungal 28S nucleicacid sequence. This specificity is essential for identifying a givenspecies of fungus in clinical specimens containing mixed fungalorganisms with a high level of reliability. The 39 organisms listed inthese Tables represent a majority of organisms that are commonlyisolated from clinical samples. While we have developed 21 speciesspecific probes (SEQ ID NO: 3 to SEQ ID NO: 23) that identify a total of19 individual organisms, the additional organisms listed in the testpanel were used to ensure that our probes did not have anycross-reactivity with other fungi likely to be present in a clinicalspecimen. The ability to accurately and reliably diagnose, and identifyto a species level, this large a number of pathogens is unmatched by anyother report. The fact that we can achieve this by probing DNA amplifiedby a single pair of "Universal" probes (SEQ ID NO: 1 and SEQ ID NO: 2)is highly advantageous as it saves time, money and effort by providingthe ability to test a single amplified target with 21 different probes(SEQ ID NO: 3 to SEQ ID NO: 23).

A GenBank search was carried out with all probes listed in SEQ ID NO: 3to SEQ ID NO: 23 in order to determine whether similar gene sequenceswere present in the database. 28S sequences for Candida albicans andCryptococcus neoformans are already present in GenBank, and as expected,the probes for Candida albicans and Cyptococcus neoformans correctlyidentified the 28S sequences from these two organsims. Ten other probesalso matched DNA sequences from a variety of genes not related to the28S gene (Table 7). This was expected because short stretches ofsequence identity can often be found for any query sequence in unrelatedgenes from the same or a different organism. This observation is knownto those versed in this art. In all cases, sequences that matched aprobe sequence were not located within the 28S rRNA genes. Our probesare used to analyze 28S DNA that has been previously amplified in apolymerase chain reaction with our probes SEQ ID NO: 1 and SEQ ID NO: 2.Under stringent conditions, these two probes only amplify DNA fromfungal 28S rRNA genes. Therefore no amplified DNA from the non-28S geneslisted in Table 7 will be available for the hybridization of probes SEQID NO: 3 to SEQ ID NO: 23. The presence of related sequences in non-28S,unamplified genes will not be detected and will, thus, not have anyeffect on the sensitivity or the specificity of our detection andidentification strategy.

                                      TABLE 3                                     __________________________________________________________________________    Detection of species specific 28S sequence with probes SEQ ID NO: 3 to        SEQ ID NO: 8                                                                  FUNGUS     SEQ ID:3                                                                           SEQ ID:4                                                                           SEQ ID:5                                                                           SEQ ID:6                                                                           SEQ ID:7                                                                           SEQ ID:8                                  __________________________________________________________________________    Acremonium sp.                                                                           -    -    -    -    -    -                                         Aspergillus clavatus                                                                     -    -    -    -    -    -                                         Aspergillus flavus                                                                       -    -    -    -    -    -                                         Aspergillus fumigatus                                                                    +    -    -    -    -    -                                         Aspergillus glaucus                                                                      -    -    -    -    -    -                                         Aspergillus nidulans                                                                     -    -    -    -    -    -                                         Aspergillus niger                                                                        -    -    -    -                                                   Aspergillus ochraceus                                                                    -    -    -    -                                                   Aspergillus terreus                                                                      -    -    -    -    -    -                                         Aspergillus unguis                                                                       -    -    -    -    -    -                                         Aspergillus ustus                                                                        -    -    -    -    -    -                                         Aspergillus sp.                                                                          -    -    -    -    -    -                                         Beauvaria sp.                                                                            -    -    -    -    -    -                                         Bipolaris sp.                                                                            -    -    -    -    -    -                                         Blastomyces dermatitidis                                                                 -    +    -    -    -    -                                         Candida albicans                                                                         -    -    -    +    -    -                                         Candida glabrata                                                                         -    -    -    -    -    -                                         Candida guilliermondii                                                                   -    -    -    -    -    -                                         Candida kefyr                                                                            -    -    -    -                                                   Candida krusei                                                                           -    -    -    -    -    -                                         Candida lusitaniae                                                                       -    -    -    -    -    -                                         Candida parapsilosis                                                                     -    -    -    -    -    -                                         Candida tropicalis                                                                       -    -    -    -    -    -                                         Chrysosporium sp.                                                                        -    -    -    -    -    -                                         Cladosporium sp.                                                                         -    -    -    -    -    -                                         Coccidioides immitis                                                                     -    -    -    +    -    -                                         Cryptococcus                                                                             -    -    -    -    +    +                                         neoformans                                                                    Curvularia sp.                                                                           -    -    -    -    -    -                                         Fusarium sp.                                                                             -    -    -    -    -    -                                         Geotrichum sp.                                                                           -    -    -    -    -    -                                         Histoplasma capsulatum                                                                   -    -    -    -    -                                              Mucor sp.  -    -    -    -    -    -                                         Penicillium sp.                                                                          -    -    -    -    -    -                                         Pseudallescheria boydii                                                                  -    -    -    -    -    -                                         Rhizopus sp.                                                                             -    -    -    -    -    -                                         Saccharomyces                                                                            -    -    -    -    -    -                                         cerevisiae                                                                    Scopulariopsis                                                                           -    -    -    -    -    -                                         brevicaulis                                                                   Sporothrix schenckii                                                                     -    -    -    -    -    -                                         Trichosporan beigelii                                                                    -    -    -    -    -    -                                         __________________________________________________________________________     + Positive                                                                    - Negative after 20 minute wash in 2X SSC                                

                                      TABLE 4                                     __________________________________________________________________________    Detection of species specific 28S sequence with probes SEQ ID NO: 9 to        SEQ ID NO: 14                                                                 FUNGUS     SEQ ID 9                                                                           SEQ ID: 10                                                                          SEQ ID: 11                                                                          SEQ ID: 12                                                                          SEQ ID: 13                                                                          SEQ ID: 14                            __________________________________________________________________________    Acremonium sp.                                                                           -    -     -     -     -     -                                     Aspergillus clavatus                                                                     -    -     -     -     -     -                                     Aspergillus flavus                                                                       -    -     -     -     -     -                                     Aspergillus fumigatus                                                                    -    -     -     -     -     -                                     Aspergillus glaucus                                                                      -    +     -     -     -     -                                     Aspergillus nidulans                                                                     -    -     -     -     -     -                                     Aspergillus niger                                                                        -    -     +     -     -     -                                     Aspergillus ochraceus                                                                    -    -     -     -     -     -                                     Aspergillus terreus                                                                      -    -     -     +     -     -                                     Aspergillus unguis                                                                       -    -     -     -     -     -                                     Aspergillus ustus                                                                        -    -     -     -     -     -                                     Aspergillus sp.                                                                          -    -     -     -     -     -                                     Beauvaria sp.                                                                            -    -     -     -     -     -                                     Bipolaris sp.                                                                            -    -     -     -     -     -                                     Blastomyces dermatitidis                                                                 -    -     -     -     -     -                                     Candida albicans                                                                         -    -     -     -     -     -                                     Candida glabrata                                                                         -    -     -     -     +     -                                     Candida guilliermondii                                                                   -    -     -     -     -     +                                     Candida kefyr                                                                            -    -     -     -     -     -                                     Candida krusei                                                                           -    -     -     -     -     -                                     Candida lusitaniae                                                                       -    -     -     -     -     -                                     Candida parapsilosis                                                                     -    -     -     -     -     -                                     Candida tropicalis                                                                       -    -     -     -     -     -                                     Chrysosporium sp.                                                                        -    -     -     -     -     -                                     Cladosporium sp.                                                                         -    -     -     -     -     -                                     Coccidioides immitis                                                                     -    -     -     -     -     -                                     Cryptococcus                                                                             -    -     -     -     -     -                                     neoformans                                                                    Curvularia sp.                                                                           -    -     -     -     -     -                                     Fusarium sp.                                                                             -    -     -     -     -     -                                     Geotrichum sp.                                                                           -    -     -     -     -     -                                     Histoplasma capsulatum                                                                   +    -     -     -     -     -                                     Mucor sp.  -    -     -     -     -     -                                     Penicillium sp.                                                                          -    -     -     -     -     -                                     Pseudallescheria boydii                                                                  -    -     -     -     -     -                                     Rhizopus sp.                                                                             -    -     -     -     -     -                                     Saccharomyces                                                                            -    -     -     -     -     -                                     cerevisiae                                                                    Scopulariopsis                                                                           -    -     -     -     -     -                                     brevicaulis                                                                   Sporothrix schenckii                                                                     -    -     -     -     -     -                                     Trichosporan beigelii                                                                    -    -     -     -     -     -                                     __________________________________________________________________________     + Positive                                                                    - Negative after 20 minute wash in 2X SSC                                

                                      TABLE 5                                     __________________________________________________________________________    Detection of species specific 28S sequence with probes SEQ ID NO: 15 to       SEQ ID NO: 20                                                                 FUNGUS     SEQ ID: 15                                                                          SEQ ID: 16                                                                          SEQ ID: 17                                                                          SEQ ID: 18                                                                          SEQ ID: 19                                                                          SEQ ID: 20                           __________________________________________________________________________    Acremonium sp.                                                                           -     -     -     -     -     -                                    Aspergillus clavatus                                                                     -     -     -     -     -     -                                    Aspergillus flavus                                                                       -     -     -     -     -     -                                    Aspergillus fumigatus                                                                    -     -     -     -     -     -                                    Aspergillus glaucus                                                                      -     -     -     -     -     -                                    Aspergillus nidulans                                                                     -     -     -     -     -     -                                    Aspergillus niger                                                                        -     -     -     -     -     -                                    Aspergillus ochraceus                                                                    -     -     -     -     -     -                                    Aspergillus terreus                                                                      -     -     -     -     -     -                                    Aspergillus unguis                                                                       -     -     -     -     -     -                                    Aspergillus ustus                                                                        -     -     -     -     -     -                                    Aspergillus sp.                                                                          -     -     -     -     -     -                                    Beauvaria sp.                                                                            -     -     -     -     -     -                                    Bipolaris sp.                                                                            -     -     -     -     -     -                                    Blastomyces dermatitidis                                                                 -     -     -     -     -     -                                    Candida albicans                                                                         -     -     -     -     -     -                                    Candida glabrata                                                                         -     -     -     -     -     -                                    Candida guilliermondii                                                                   -     -     -     -     -     -                                    Candida kefyr                                                                            +     -     -     -     -     -                                    Candida krusei                                                                           -     +     -     -     -     -                                    Candida lusitaniae                                                                       -     -     +     -     -     -                                    Candida parapsilosis                                                                     -     -     -     +     -     -                                    Candida tropicalis                                                                       -     -     -     -     +     -                                    Chrysosporium sp.                                                                        -     -     -     -     -     -                                    Cladosporium sp.                                                                         -     -     -     -     -     -                                    Coccidioides immitis                                                                     -     -     -     -     -     -                                    Cryptococcus                                                                             -     -     -     -     -     -                                    neoformans                                                                    Curvularia sp.                                                                           -     -     -     -     -     -                                    Fusarium sp.                                                                             -     -     -     -     -     -                                    Geotrichum sp.                                                                           -     -     -     -     -     -                                    Histoplasma capsulatum                                                                   -     -     -     -     -     -                                    Mucor sp.  -     -     -     -     -     -                                    Penicillium sp.                                                                          -     -     -     -     -     -                                    Pseudallescheria boydii                                                                  -     -     -     -     -     -                                    Rhizopus sp.                                                                             -     -     -     -     -     -                                    Saccharomyces                                                                            -     -     -     -     -     -                                    cerevisiae                                                                    Scopulariopsis                                                                           -     -     -     -     -     -                                    brevicaulis                                                                   Sporothrix schenckii                                                                     -     -     -     -     -     -                                    Trichosporan beigelii                                                                    -     -     -     -     -     -                                    __________________________________________________________________________     + Positive                                                                    - Negative after 20 minute wash in 2X SSC                                

                  TABLE 6                                                         ______________________________________                                        Detection of species specific 28S sequence with probes SEQ ID NO: 21 to       SEQ ID NO: 23                                                                 FUNGUS        SEQ ID: 21 SEQ ID: 22                                                                              SEQ ID: 23                                 ______________________________________                                        Acremonium sp.                                                                              -          -         -                                          Aspergillus clavatus                                                                        -          -         -                                          Aspergillus flavus                                                                          +          -         -                                          Aspergillus fumigatus                                                                       -          -         -                                          Aspergillus glaucus                                                                         -          -         -                                          Aspergillus nidulans                                                                        -          -         -                                          Aspergillus niger                                                                           -          -         -                                          Aspergillus ochraceus                                                                       -          -         -                                          Aspergillus terreus                                                                         -          -         -                                          Aspergiilus unguis                                                                          -          -         -                                          Aspergillus ustus                                                                           -          -         -                                          Aspergillus sp.                                                                             -          -         -                                          Beauvaria sp. -          -         -                                          Bipolaris sp. -          -         -                                          Blastomyces dermatitidis                                                                    -          -         -                                          Candida albicans                                                                            -          -         -                                          Candida glabrata                                                                            -          -         -                                          Candida guilliermondii                                                                      -          -         -                                          Candida kefyr -          -         -                                          Candida krusei                                                                              -          -         -                                          Candida lusitaniae                                                                          -          -         -                                          Candida parapsilosis                                                                        -          -         -                                          Candida tropicalis                                                                          -          -         -                                          Chrysosporium sp.                                                                           -          -         -                                          Cladosporium sp.                                                                            -          -         -                                          Coccidioides immitis                                                                        -          -         -                                          Cryptococcus neoformans                                                                     -          -         -                                          Curvularia sp.                                                                              -          -         -                                          Fusarium sp.  -          -         -                                          Geotrichum sp.                                                                              -          -         -                                          Histoplasma capsulatum                                                                      -          -         -                                          Mucor sp.     -          -         -                                          Penicillium sp.                                                                             -          -         -                                          Pseudallescheria boydii                                                                     -          -         -                                          Rhizopus sp.  -          -         -                                          Saccharomyces cerevisiae                                                                    -          -         -                                          Scopulariopsis brevicaulis                                                                  -          -         -                                          Sporothrix schenckii                                                                        -          +         +                                          Trichosporon beigelii                                                                       -          -         -                                          ______________________________________                                         +Positive                                                                     -Negative after 20 minute wash in 2X SSC                                 

                                      TABLE 7                                     __________________________________________________________________________    GenBank search results listing genes from other organisms having 100%         identity to probes SEQ ID NO: 3 to SEQ ID NO: 23                                          PROBE          GENE                                                           SEQ ID                                                                            ORGANISM   MATCHED*                                                                              ACCESSION                                              NO: MATCHED    (see note below)                                                                      NUMBER                                     __________________________________________________________________________    Aspergillus fumigatus                                                                     3   --         --      --                                         Blastomyces dermatitidis                                                                  4   Streptomyces verticillus                                                                 bleomycin acetyl                                                                      L26955                                                                transferase                                                    4   Giardia muris                                                                            upstream of                                                                           X65063, S53320                                                        rRNA genes                                                     4   Aspergillus nidulans                                                                     uric acid-xanthine                                                                    X71807                                                                permease                                                       4   Homo sapiens                                                                             T-cell surface                                                                        X16996                                                                glycoprotein                                                   4   Homo sapiens                                                                             MIC2    M16279, M22557,                                                               J03841, M22556                             Candida albicans                                                                          5   Candida albicans                                                                         28S rRNA                                                                              L28817                                     Coccidioides immitis                                                                      6   --         --      --                                         Cryptococcus neoformans                                                                   7   Cryptococcus                                                                             28S rRNA                                                                              L14067,L14068,                                             neoformans                                                    Cryptococcus neoformans                                                                   8   Cryptococcus                                                                             28S rRNA                                                                              L14067, L14068,                                            neoformans                                                                                       L20964                                                 8   Escherichia coli                                                                         0111 cld                                                                              Z17241                                     Histoplasma capsulatum                                                                    9   --         --      --                                         Aspergillus glaucus                                                                       10  Pseudomonas                                                                              cob genes                                                                             M62866                                                     denitrificans                                                 Aspergillus niger                                                                         11  --         --      --                                         Aspergillus terreus                                                                       12  Human cytomegalovirus                                                                    genome  X17403                                                 12  Homo sapiens                                                                             GABA receptor                                                                         L08485                                     Candida glabrata                                                                          13  Homo sapiens                                                                             Class 1 MHC                                                                           X03664, X03665                             Candida guilliermondii                                                                    14  --         --      --                                         Candida kefyr                                                                             15  --         --      --                                         Candida krusei                                                                            16  Pseudomonas syringae                                                                     penicillin binding                                                                    L28837                                                                protein                                            Candida lustaniae                                                                         17  Chicken    AK1     D00251                                                 17  Mouse      IL10    M84340                                     Candida parapsilosis                                                                      18  Polytomella agilis                                                                       beta-2 tubulin                                                                        M33373                                                 18  Tobacco chloroplast                                                                      genome  Z00044, S54304                                         18  Aedes aegypti                                                                            amylase L03640                                                 18  Homo sapians                                                                             chromosome                                                                            L14473                                                                13q14                                              Candida tropicalis                                                                        19  --         --      --                                         Pseudallescheria boydii                                                                   20  Drosophila melanogaster                                                                  AcTr66B X71789                                                     Cow        actin 2 D12816                                     Aspergillus flavus                                                                        21  --         --      --                                         Sporothrix schenckii                                                                      22  --         --      --                                         Sporothrix schenckii                                                                      23  Sulfate reducing bacteria                                                                FMN binding                                                                           D21804                                                                protein                                                        23  Equine herpesvirus 1                                                                     genome  M86664                                     __________________________________________________________________________     *Note:                                                                        As discussed earlier in this document, the presence of sequences similar      to probes SEQ ID NO:3 to SEQ ID NO: 23 in genes not related to 28S does       not have any effect on the specificity or sensitivity of our diagnostic       strategy. Our species specific probes are used to analyze 28S DNA that ha     been previously amplified in a polymerase chain reaction with our probes      SEQ ID NO: 1 and SEQ ID NO:2. These two probes will not amplify DNA from      any gene other than 28S in column #4(GENE MATCHED), and therefore no          amplified DNA from these non28S genes will be available for the               hybridization of probes SEQ ID NO: 3 to SEQ ID NO: 23.                   

EXAMPLE 2

Use of method in example 1 to test clinical specimens for specificfungal organisms.

Clinical samples taken from the respiratory and gastrointestinal tractof healthy individuals almost always contain fungal flora. Most of thesefungi are non-pathogenic, but may give false positives on traditionalimmunochemical diagnostic tests for pathogenic fungi.

We obtained 44 specimens from diverse sources ranging from sputum andincision drainage tubes, to intervertebral disc and lung biopsies.Traditional smear and culture results showed that all 44 contained atleast 1 type of fungus. In order to test the efficacy of our probes, weextracted DNA from all 44 clinical samples and used probes SEQ ID NO: 1& 2 in a polymerase chain reaction to amplify fungal 28S sequencespresent in these samples.

DNA was extracted from all clinical samples by our modification of thetechnique of Chomczynski and Sacchi which originally described the useof acid guanidinium thiocyanate-phenol-chloroform to preferentiallyextract RNA from cells and tissues. We replaced room temperature celllysis by boiling lysis, and acid guanidiniumthiocyanate-phenol-chloroform extraction by alkaline phenol-guanidinethiocyanate to preferentially extract DNA from cells. 1.5 ml Sarsted(Newton, N.C.) polypropylene screw cap tubes with o-ring seals were usedfor the extractions. 200 ul of specimen was added to 500 ul of GPTreagent (6M guanidine thiocyanate dissolved in 50 mM tris pH 8.3 mixedwith an equal volume of phenol buffered in tris pH 8.0). This was mixedby vortexing and immediately placed in a boiling water bath for 15minutes. The tubes were spun in a microcentrifuge for 5 seconds and 250ul of chloroform/isoamyl alcohol (24:1 by volume) was added and mixed byvortexing. The liquid phases were separated by centrifugation for 10minutes and 450 ul of aqueous (upper) phase was transferred to a freshtube. The aqueous phase was mixed with 500 ul of 100% isopropanol andplaced at -20° C. for at least 1 hour. At the end of this period thetubes were centrifuged for 15 minutes and the supernatant removedwithout disturbing the nucleic acid pellet. The pellet was washed with500 ul of ice-cold 70% ethanol to remove traces of GPT reagent by gentlyinverting 2 times and then centrifuged for 5 minutes. The ethanol wasremoved and the pellet dried in a speed vac for 10 minutes. The pelletwas resuspended in 25 ul of sterile deionized water and 5 ul was used ina 50 ul PCR amplification. The PCR was carried out as a hot-startreaction using the thermal cycling conditions for probes SEQ ID NO: 1and SEQ ID NO: 2 described in example 1. Gel electrophoresis showed thatprobes SEQ ID NO: 1 and SEQ ID NO: 2 successfully amplified DNA from all44 specimens.

The amplified DNA from each specimen was transferred to a positivelycharged polysulphone based membrane. We radioactively labeled ourspecies specific probes SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQID NO: 7, and sequentially probed the membrane to test for the presenceof 28S rDNA from Aspergillus fumigatus, Candida albicans, Coccidioidesimmitis and Cryptococcus neoformans respectively. Membrane blocking,probe hybridization and washes were done exactly as described inexample 1. The results are shown in Table 8.

No false positives were observed, indicating a specificity of 100% forthese 4 probes in the clinical specimens tested. 10 out of 12 culturepositive samples for Aspergillus fumigatus, and 11 out of 13 samples ofCandida albicans were identified, indicating a detection sensitivity ofabout 85% for these two probes. Additionally, two out of twoCoccidioides immitis and two out of two Cryptococcus neoformans werecorrectly identified (detection sensitivity of 100%). As seen by theseresults, the probes described in this invention can be used on a diversevariety of clinical specimens with excellent efficacy.

                                      TABLE 8                                     __________________________________________________________________________    Detection of Aspergillus fumigatus, Candida albicans, Coccidioides            immitis and                                                                   Cryptococcus neoformans in clinical specimens using species specific          probes.                                                                                 Smear and culture                                                                      PCR with                                                   Specimen type                                                                           results  SEQ ID: 1, 2                                                                        SEQ ID: 3                                                                          SEQ ID: 5                                                                          SEQ ID: 6                                                                          SEQ ID: 7                             __________________________________________________________________________    U035 sputum                                                                             A. flavus                                                                              +     -    -    -    -                                     U069 pleura                                                                             A. fumigatus                                                                           +     +    -    -    -                                     U070 bronchial wash                                                                     A. flavus                                                                              +     -    -    -    -                                     M019 bronchial wash                                                                     A. fumigatus                                                                           +     +    -    -    -                                     M020 sputum                                                                             mixed fungal flora                                                                     +     -    +    -    -                                     X35254 sputum                                                                           C. albicans                                                                            +     -    +    -    -                                     M20910 sputum                                                                           A. fumigatus                                                                           +     +    -    -    -                                     M055 sputum                                                                             C. albicans                                                                            +     -    +    -    -                                     M056 abdominal                                                                          mixed fungal flora                                                                     +     -    -    -    -                                     M057 drainage tube                                                                      C. albicans                                                                            +     -    (--) -    -                                     M059 ind. sputum                                                                        C. albicans                                                                            +     -    +    -    -                                     M060 ind. sputum                                                                        mixed fungal flora                                                                     +     -    -    -    -                                     M083 bronchial wash                                                                     C. albicans                                                                            +     -    +    -    -                                     M084 sputum                                                                             A. fumigatus                                                                           +     (--) -    -    -                                     M085 throat                                                                             C. albicans                                                                            +     -    (--) -    -                                     A001 sputum                                                                             A. fumigatus                                                                           +     (--) -    -    -                                     A002 leg  Blastomyces                                                                            +     -    -    -    -                                     A003 leg  Blastomyces                                                                            +     -    -    -    -                                     A005 disc A. fumigatus                                                                           +     +    -    -    -                                     A037 disc A. fumigatus                                                                           +     +    -    -    -                                     A039 trachea                                                                            C. albicans                                                                            +     -    +    -    -                                     A040 trachea                                                                            C. albicans                                                                            +     -    +    -    -                                     A102 empyema                                                                            A. fumigatus                                                                           +     +    -    -    -                                     Y004 sputum                                                                             C. albicans                                                                            +     -    +    -    -                                     Y016 induced sputum                                                                     Coccidioides                                                                           +     -    -    +    -                                     Y028 sputum                                                                             Coccidioides                                                                           +     -    -    +    -                                     J003 chest                                                                              Aspergillus sp.                                                                        +     -    -    -    -                                     J045 bronchial wash                                                                     C. albicans                                                                            +     -    +    -    -                                     J046 ethmoid                                                                            yeast    +     -    -    -    -                                     J047 chest                                                                              A. fumigatus                                                                           +     +    -    -    -                                     J048 sputum                                                                             C. albicans                                                                            +     -    +    -    -                                     J073 lung Aspergillus sp.                                                                        +     -    -    -    -                                     J074 lung A. fumigatus                                                                           +     +    -    -    -                                     U107 lip  A. fumigatus                                                                           +     +    -    -    -                                     U033 sputum                                                                             mixed fungal flora                                                                     +     -    -    -    -                                     U071 sputum                                                                             C. albicans                                                                            +     -    +    -    -                                     U072 BA lavage                                                                          Sporothrix                                                                             +     -    -    -    -                                     U073 knee Histoplasma                                                                            +     -    -    -    -                                     U074 mandible                                                                           Cryptococcus                                                                           +     -    -    -    +                                     U075 CSF  Cryptococcus                                                                           +     -    -    -    +                                     U076 knee Histoplasma                                                                            +     -    -    -    -                                     U077 soft tissue                                                                        Histoplasma                                                                            +     -    -    -    -                                     U051 buccal                                                                             A. fumigatus                                                                           +     +    -    -    -                                     Y055 sputum                                                                             mixed fungal flora                                                                     +     -    -    -    -                                     __________________________________________________________________________     + Positive                                                                    - Negative                                                                    (--) Missed                                                              

EXAMPLE 3.

DNA sequence based identification of unknown fungal organisms.

Another utility of our probes is in the rapid DNA sequence basedidentification of a pure culture of fungus. Probes SEQ ID NO: 1 and SEQID NO: 2 are used in a polymerase chain reaction to amplify 28S rDNAfrom an unknown fungus. Probes SEQ ID NO: 1 or SEQ ID NO: 2 are thenused as sequencing primers to obtain DNA sequence from this amplified28S DNA belonging to the unknown fungus. This DNA sequence is comparedto the fungal 28S DNA sequences in our database, and a sequence matchat, or overlapping any one of the probe sequences in SEQ ID NO: 3 to SEQID NO: 23 will confirm the identity of the fungus. This technique cannotbe used directly on clinical samples, as these usually contain DNA frommore than one fungus, and the DNA sequence generated will consist ofoverlapping sequences of several organisms. This technique has utilityin rapidly and reliably identifying colonies of a single fungus onculture plates, clinical specimens, food, pharmaceutical, environmentalor other samples containing only one species of fungus.

EXAMPLE 4.

Capture and identification of target DNA or RNA

All primers and probes described in this invention disclosure may belabeled with any detectable moiety including, but not limited toradioisotopes, enzymes, antigens, antibodies, chemiluminescent reagentsand fluorescent chemicals. Additionally, these probes may be modifiedwithout changing the substance of their purpose by terminal addition ofnucleotides designed to incorporate restriction sites or other usefulsequences. These probes may also be modified by the addition of moieties(including, but not limited to para-magnetic particles, biotin,fluorescein, dioxigenin, antigens, antibodies) or attached to the wallsof microtiter trays to assist in the solid phase capture andpurification of these probes and any DNA or RNA hybridized to theseprobes. Fluorescein may be used as a signal moiety as well as a capturemoiety, the latter by interacting with an anti-fluorescein antibody.

A typical utility of these modifications would be as follows. PrimersSEQ ID NO: 1 and SEQ ID NO: 2 would be utilized to amplify 28S rDNA froma sample, if present, as described previously. Primers would be modifiedso as to contain a biotin moiety at their 5' ends. A streptavidin solidphase, such as a paramagnetic particle, would be used to separate PCRproducts, if present, from the reaction mixture. The amplified targetmay be subsequently hybridized to a third probe ((SEQ ID NO: 3) to (SEQID NO: 23) or their complements) attached to a detectable moiety todetermine which species of fungus is present in the given sample.Multiple probes, each labeled with a different detectable moiety may beused at one time to analyze the amplified target.

Alternatively, Primers SEQ ID NO: 1 and SEQ ID NO: 2 would be utilizedto amplify 28S rDNA from a sample, if present, as above. In a separatereaction, individually, either SEQ ID NO: 1 or SEQ ID NO: 2 would bemodified by attachment to a solid phase capture moiety, such as aparamagnetic particle, and SEQ ID NO: 3 to SEQ ID NO: 23 (or theircomplements) would be modified by addition of a detectable moiety.Alternately, in the amplicon, any sequences delimited by SEQ ID NO: 1and SEQ ID NO: 2 with the exception of the SEQ ID NO: 3 to SEQ ID NO:23, may be used in the design of a capture probe. One of the probesattached to a solid phase (SEQ ID NO: 1 and SEQ ID NO: 2) or any otherappropriately designed sequences and one of the probes modified byattachment to a detectable moiety (SEQ ID NO: 3 to SEQ ID NO: 23 ortheir complements) would be hybridized together, in solution, toproducts of the PCR, if they had been generated. The hybrids, ifpresent, would be captured from the solution, and analyzed by a methodappropriate to the detection moiety. Detection of the hybridized probewould indicate which species of fungus was present in the given sample.Multiple probes, each labeled with a different detectable moiety may beused at one time to analyze the amplified target.

EXAMPLE 5.

Species-specific amplification of fungal DNA

Another utility of the probes described in this invention is their usageas primers in the direct detection of a specific fungal species byvirtue of a nucleic acid amplification reaction. In this embodiment, oneprimer is a universal one, such as (SEQ ID NO: 1) or (SEQ ID NO:2), andthe other is a species-specific primer selected from the groupconsisting of (SEQ ID NO:3) to (SEQ ID NO: 23) or the complementsthereof. One variation of this approach is the substitution of (SEQ IDNO: 1) or (SEQ ID NO:2) with any functional sequence located inproximity to the species-specific primer.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 25                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for fungal organisms                   (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       GTGAAATTGTTGAAAGGGAA20                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for fungal organisms                   (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       GACTCCTTGGTCCGTGTT18                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus fumigatus              (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       CTCGGAATGTATCA14                                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Blastomyces dermatitidis           (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       ACTCCCCCACGGG13                                                               (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida albicans                   (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       CCTCTGACGATGCT14                                                              (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Coccidioides immitis               (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       TCTGGCGGTTGGTT14                                                              (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Cryptococcus neoformans            (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       CTCCTGTCGCATAC14                                                              (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Cryptococcus neoformans            (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       AGTTCTGATCGGTG14                                                              (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Histoplasma capsulatum             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       CAATCCCCCGCGGC14                                                              (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus glaucus                (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      GTGTCATGCGGCCA14                                                              (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus niger                  (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 11:                                      CCCTGGAATGTAGT14                                                              (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus terreus                (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 12:                                      GCTTCGGCCCGGTG14                                                              (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida glabrata                   (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 13:                                      CTTGGGACTCTCGC14                                                              (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida guilliermondii             (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 14:                                      ATATTTTGTGAGCC14                                                              (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida kefyr                      (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 15:                                      TTCGGCTTTCGCTG14                                                              (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida krusei                     (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 16:                                      GGGATTGCGCACCG14                                                              (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida lusitaniae                 (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 17:                                      GCCTCCATCCCTTT14                                                              (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida parapsilosis               (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 18:                                      ATAAGTGCAAAGAA14                                                              (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Candida tropicalis                 (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 19:                                      AGAATTGCGTTGGA14                                                              (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Pseudallescheria boydii            (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 20:                                      GCGATGGGAATGTG14                                                              (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Aspergillus flavus                 (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 21:                                      AGACTCGCCTCCAG14                                                              (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Sporothrix schenckii               (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 22:                                      CGGACCACCCGGCG14                                                              (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic acid probe for Sporothrix schenckii               (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 23:                                      CGGCGGCATGCCCC14                                                              (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic Acid Primer                                       (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 24:                                      ATCAATAAGCGGAGGAAAAG20                                                        (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: Nucleic Acid Primer                                       (iii) HYPOTHETICAL: No                                                        (iv) ANTI-SENSE: No                                                           (v) SEQUENCE DESCRIPTION: SEQ ID NO: 25:                                      CTCTGGCTTCACCCTATTC19                                                         __________________________________________________________________________

We claim:
 1. A method of determining whether one or more fungal speciesselected from the group of fungal species consisting of Aspergillusfumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioidesimmitis, Cryptococcus neoformans, Histoplasma capsulatum, Aspergillusglaucus, Aspergillus niger, Aspergillus terreus, Candida glabrata,Candida guilliermindii, Candida kefyr, Candida krusei, Candidalusitaniae, Candida parapsilosis, Candida tropicalis, Pseudallescheriaboydii, Asperillus flavus and Sporothrix schenckii is present in asample of fungi, said method comprising the following steps:a)extracting nucleic acid material from fungi contained in said sample; b)adding two known oligonucleocide primers, one of said primers being (SEQID NO:1) or (SEQ ID NO:2), said primers bracketing a hypervariableregion on the 28S rDNA or rRNA present in the fungal species of saidgroup; c) amplifying the sequence between said primers; and d) using oneor more labeled probes directed to a portion of the hypervariable regionbracketed by said primers, each said labeled probe being specific forone of said fungal species from said group, to determine whether saidfungal species identified by each said labeled probe is present in saidsample.
 2. The method of claim 1 in which, in said amplifying step, saidamplifying procedure is the polymerase chain reaction.
 3. The method ofclaim 1 in which said one or more probes is selected from the groupconsisting of (SEQ ID NO:3), (SEQ ID NO:4), (SEQ ID NO:5), (SEQ IDNO:6), (SEQ ID NO:7), (SEQ ID NO:8), (SEQ ID NO:9), (SEQ ID NO:10), (SEQID NO:11), (SEQ ID NO:12), (SEQ ID NO:13), (SEQ ID NO:14), (SEQ IDNO:15), (SEQ ID NO:16), (SEQ ID NO:17), (SEQ ID NO:18), (SEQ ID NO:19),(SEQ ID NO:20), (SEQ ID NO:21), (SEQ ID NO:22) and (SEQ ID NO:23). 4.The method of claim 1 wherein, in step (d), more than one probe is used,each said probe being connected to (a) a different signal moiety or (b)a moiety which allows separation of said probes.
 5. A method ofdetermining whether one or more fungal species selected from the groupof fungal species consisting of Aspergillus fumigatus, Blastomycesdermatitidis, Candida albicans, Coccidioides immitis, Cryptococcusneoformans, Histoplasma capsulatum, Aspergillus glaucus, Aspergillusniger, Aspergillus terreus, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis,Candida tropicalis, Pseudallescheria boydii, Aspergillus flavus andSporothrix schenckii is present in a sample of fungi, said methodcomprising the following steps:a) extracting nucleic acid material fromfungi contained in said sample; b) adding a universal fungal probeselected from the group consisting of (SEQ ID NO: 1), (SEQ ID NO:2) andthe complements thereof; c) using one or more second probes, each saidsecond probe being specific for one of said fungal species from saidgroup, wherein said one or more second probes each has a nucleotideresidue sequence selected from the group consisting of (SEQ ID NO:3),(SEQ ID NO:4), (SEQ ID NO:5), (SEQ ID NO:6), (SEQ ID NO:7), (SEQ IDNO:8), (SEQ ID NO:9), (SEQ ID NO:10), (SEQ ID NO:11), (SEQ ID NO:12),(SEQ ID NO:13), (SEQ ID NO:14), (SEQ ID NO:15), (SEQ ID NO:16), (SEQ IDNO:17), (SEQ ID NO:18), (SEQ ID NO:19), (SEQ ID NO:20), (SEQ ID NO:21),(SEQ ID NO:22), (SEQ ID NO:23) and the complements thereof; and d)determining whether said fungal species identified by each said secondprobe is present in said sample,wherein at least one of said probes isconnected to a signal moiety and at least one of said probes isconnected to a moiety that allows separation of said probes.
 6. A methodof determining whether one or more fungal species selected from thegroup of fungal species consisting of Aspergillus fumigatus, Blastomycesdermatitidis, Candida albicans, Coccidioides immitis, Cryptococcusneoformans, Histoplasma capsulatum, Aspergillus glaucus, Aspergillusniger, Aspergillus terreus, Candida glabrata, Candida guilliermondii,Candida kefyr, Candida krudei, Candida lusitaniae, Candida parapsilosis,Candida tropicalis, Pseudallescheria boydii, Aspergillus flavus andSporothrix schenckii is present in a sample of fungi, said methodcomprising the following steps:a) extracting nucleic acid material fromfungi contained in said sample; and b) using one or more labeled probes,each said labeled probe being specific for one of said fungal speciesfrom said group, to determine whether said fungal species identified byeach said labeled probe is present in said sample, wherein said one ormore labeled probes each has a nucleotide residue sequence selected fromthe group consisting of (SEQ ID NO:3), (SEQ ID NO:4), (SEQ ID NO:5),(SEQ ID NO:6), (SEQ ID NO:7), (SEQ ID NO:8), (SEQ ID NO:9), (SEQ IDNO:10), (SEQ ID NO:11), (SEQ ID NO:12), (SEQ ID NO:13), (SEQ ID NO:14),(SEQ ID NO:15), (SEQ ID NO:16), (SEQ ID NO:17), (SEQ ID NO:18), (SEQ IDNO:19), (SEQ ID NO:20), (SEQ ID NO:21), (SEQ ID NO:22), (SEQ ID NO:23)and the complements thereof.
 7. An oligonucleotide probe/primer forfungi, said probe/primer having the nucleotide residue sequence of (SEQID NO:1) or the complement thereof.
 8. An oligonucleotide probe/primerfor fungi, said probe/primer having the nucleotide residue sequence of(SEQ ID NO:2) or the complement thereof.
 9. An oligonucleotidehybridization probe for Aspergillus fumigatus, said probe having thenucleotide residue sequence of (SEQ ID NO:3) or the complement thereof.10. An oligonucleotide hybridization probe for Blascomyces dermatitidis,said probe having the nucleotide residue sequence of (SEQ ID NO:4) orthe complement thereof.
 11. An oligonucleotide hybridization probe forCandida albicans, said probe having the nucleotide residue sequence of(SEQ ID NO:5) or the complement thereof.
 12. An oligonucleotidehybridization probe for Coccidioides immitis, said probe having thenucleotide residue sequence of (SEQ ID NO:6) or the complement thereof.13. An oligonucleocide hybridization probe for Cryptococcus neoformans,said probe having the nucleotide residue sequence of (SEQ ID NO:7) orthe complement thereof.
 14. An oligonucleotide hybridization probe forCryptococcus neoformans, said probe having the nucleotide residuesequence (SEQ ID NO:8) or the complement thereof.
 15. An oligonucleotidehybridization probe for Hictoplasma capsulatum, said probe having thenucleotide residue sequence of (SEQ ID NO:9) or the complement thereof.16. An oligonucleotide hybridization probe for Aspergillus glaucus, saidprobe having the nucleotide residue sequence of (SEQ ID NO:10) or thecomplement thereof.
 17. An oligonucleotide hybridization probe forAspergillus niger, said probe having the nucleotide residue sequence of(SEQ ID NO:11) or the complement thereof.
 18. An oligonucleotidehybridization probe for Aspergillus terreus, said probe having thenucleotide residue sequence of (SEQ ID NO:12) or the complement thereof.19. An oligonucleotide hybridization probe for Candida glabrata, saidprobe having the nucleotide residue sequence of (SEQ ID NO:13) or thecomplement thereof.
 20. An oligonucleotide hybridization probe forCandida guilliermondii, said probe having the nucleotide residuesequence of (SEQ ID NO:14) or the complement thereof.
 21. Anoligonucleotide hybridization probe for Candida kefyr, said probe havingthe nucleotide residue sequence of (SEQ ID NO:15) or the complementthereof.
 22. An oligonucleotide hybridization probe for Candida krusei,said probe having the nucleotide residue sequence (SEQ ID NO:16) or thecomplement thereof.
 23. An oligonucleotide hybridization probe forCandida lusitaniae, said probe having the nucleotide residual sequenceof (SEQ ID NO 17) or the complement thereof.
 24. An oligonucleotidehybridization probe for Candida parapsilosis, said probe having thenucleotide residue sequence of (SEQ ID NO:18) or the complement thereof.25. An oligonucleotide hybridization probe for Candida tropicalis, saidprobe having the nucleotide residue sequence of (SEQ ID NO:19) or thecomplement thereof.
 26. An oligonucleocide hybridization probe forPseudallescheria boydii, said probe having the nucleotide residuesequence of (SEQ ID NO:20) or the complement thereof.
 27. Anoligonucleotide hybridization probe for Aspergillus flavus, said probehaving the nucleotide residue sequence of (SEQ ID NO:21) or thecomplement thereof.
 28. An oligonucleotide hybridization probe forSporothrix schenckii, said probe having the nucleotide residue sequenceof (SEQ ID NO:22) or the complement thereof.
 29. An oligonucleotidehybridization probe for Sporothrix schenckii, said probe having thenucleotide residue sequence of (SEQ ID NO:23) or the complement thereof.